Telomeres are specialized structures at chromosome ends, which consist of tandemly repeated DNA sequences and associated proteins (König and Rhodes, Trends Biochem. Sci., 22: 43-47, 1997). In normal human somatic cells, telomeric DNA progressively shortens with each cell division. Critically short telomeres are thought to cause irreversible cell growth arrest and cellular senescence (Autexier and Greider, Trends Biochem. Sci., 21: 387-391, 1996). In contrast, most cancer cells have mechanisms that compensate for telomere shortening, which allow them to stably maintain their telomeres and grow indefinitely (Chiu and Harley, Proc. Soc. Exp. Biol. Med., 214: 99-106, 1997; Autexier and Greider, Trends Biochem. Sci., 21: 387-391, 1996; Bodnar et al., Science, 279: 349-352, 1998).
Telomerase is a specialized, multi-subunit DNA polymerase responsible for the replication of telomeres. Thus, telomerase compensates for telomere shortening in cells where telomerase is active. Telomerase is highly active in many immortalized cell lines and human cancers, and is thought to be a factor in their continuing ability to replicate. In contrast, telomerase activity is low or absent in most normal somatic cells, which is thought to be a factor in their limited ability to replicate.
Several components of the human telomerase complex have been identified. Of these, the RNA component, which acts as an intrinsic template for telomeric repeat synthesis, and the telomerase catalytic subunit, known as human telomerase reverse transcriptase (hTERT), are necessary and sufficient for telomerase activity in vitro (Masutomi et al., J. Biol. Chem., 275: 22568-22573, 2000).
hTERT expression at the mRNA level is correlated with human telomerase activity. Accordingly, the hTERT gene is highly expressed in many immortalized cell lines and human cancers and has limited expression in most normal somatic cells. The native regulatory regions that underlie the differential expression of the hTERT gene in cancer and normal cells have been isolated and characterized (e.g., Leem et al., Oncogene, 21(5): 769-777, 2002; Tzukerman et al., Mol. Biol. Cell, 11: 4381-4391, 2000; Wick et al., Gene, 232(1): 97-106, 1999; Horikawa et al., Can. Res., 59: 826-830, 1999; Cong et al., Hum. Mol. Genet., 8(1): 137-42, 1999). Deletion analyses of the hTERT promoter revealed that no more than several hundred base pairs located immediately upstream of the translation initiation codon were required for differential activity of the promoter in cancer and normal cells (e.g., Horikawa et al., Can. Res., 59: 826-830, 1999). Thus, relatively small fragments of the hTERT promoter may be used to drive cancer-specific expression of operably linked nucleic acid sequences.
The cancer-specific activity of the hTERT promoter makes it a candidate for anti-cancer strategies. Studies using all or part of the native hTERT promoter to drive heterologous cytotoxic gene expression have shown selective killing of cancer cells in experimental models (e.g., Majumdar et al., Gene Therapy, 8: 568-578, 2001). However, an important characteristic of any promoter-driven therapeutic strategy must be its ability to target cancer cells while leaving normal cells relatively unaffected. The native hTERT promoter is not entirely silent in normal cells. Thus, heterologous nucleic acids to which the native promoter is operably linked will be expressed in some normal cells.
Even low-level expression of a cytotoxin in normal cells may cause undesired side effects. Thus, it would be advantageous to have available artificial TERT promoters that have minimal activity in normal cells but that maintain high-level expression in other cell types, such as cancer cells; thereby enhancing the differential expression of a TERT promoter in normal and cancer cells, for example.